Part:BBa_M36532:Experience

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Applications of BBa_M36532

A. Experiments to confirm presence of plasmids in cells

Transformed Yeast Growth on SC-LEU and SC-HIS Plates

Two days after the yeast transformations, we did not observe any colony growth on either plate and put the plates back into the 30° C incubator. After nine days, the plates were removed and showed robust colony growth for both transformed constructs.

PCR and Gel Electrophoresis

PCR and subsequent gel electrophoresis results reveal that our yeast had been successfully transformed with our designed plasmids. The strong bands visible in lane (+PHO and +PHO2) of correspond to a DNA fragment of length between 700-750. The strong bands visible in lane lane (+ACR121 and +ACR122) corresponds to a DNA fragment of length between 650-700. The absence of the same bands in our negative control lanes suggests that the amplified fragment belonged to the plasmid DNA that were extracted. Given their lengths, the bands are likely to correspond to our PHO84 and ACR1 gene fragments respectively, which were expected to be 747bp and 647bp in length. This suggests that our transformed yeast contain our plasmids.

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B. Experiments that determine whether or not our designed actuator exhibits desired performance characteristics in S. cerevisiae

Cell Density Functional Assay

We conducted a functional assay with five arsenate concentrations from 12 ppb to 120,000 ppb and at five different time points for each of these two plasmid constructs. In this assay, our negative control was two replicates of transformed cells without arsenate, which demonstrates how the cells naturally grew in the 96-well plate. Two replicates of transformed cells with arsenate and without plasmid induction or repression provides a baseline of the cells’ intrinsic arsenic resistance as well as the toxicity of the plasmid itself. The two experimental conditions were transformed cells with arsenate which were repressed with 3% glucose and the plasmids induced by 3% galactose.

ACR12 plasmid

The intended function of the ACR12 gene construct is to reduce arsenate to arsenite and to confer arsenite resistance. Because this gene construct increases cell resistance, we hypothesize that I) cell density should decrease for all conditions as arsenate concentration increases II) at a given arsenate concentration, the OD600 of cells with the galactose-activated plasmid would be higher than those with the glucose-repressed plasmid and the baseline.

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As shown the graph above, at one hour, the OD600 was much higher at the higher concentrations of arsenate than the lower concentrations, challenging hypothesis I. We hypothesize that this unexpected reading could have been due to the fact that during sample preparation, we loaded the lowest concentrations of arsenate first and had a 5-7 minute difference between the time arsenate was loaded into the 12 ppb well and the 120,000 ppb well. According to Shen, arsenate uptake into cells occurs quite rapidly, often within 10 minutes; however, the ACR genes often take two or more hours to fully take effect.4 As a result, more cells at the lower concentrations could have died before we put the plate in the shaker to grow.

The data were consistent with the expected trend for hypothesis II for both control and experimental groups and at all arsenate concentrations. After 2 hours of growth, the OD600 for different arsenate concentrations started to level off, with the galactose-induced samples having a highly statistically significant (p = 1.4 *10-5) difference from the baseline. We hypothesize that this may be the timepoint where the gene is expressed and starts reducing arsenate and conferring arsenite resistance.

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By the next morning (15-20 hours), both the general trends of hypothesis I and II were supported by the data; however, the difference between the galactose-induced promoter and the baseline was no longer statistically significant (p=0.413). We hypothesize that the difference is no longer statistically significant due to the saturation of the ACR12 gene complex in converting arsenate to arsenite before 15 hours.

PHO84 Plasmid

The intended function of the PHO84 gene construct is to increase arsenate uptake into the cell. Since this gene has not been found to confer arsenate or arsenite resistance, our hypothesis was that the OD600 of the cells with the galactose-activated promoter would be lower than those with the glucose-repressed promoter. The cells with the glucose-repressed promoter should be comparable with the baseline of transformed cells with only arsenate.

However, at all concentrations and over time for all experimental groups, the observed trend was not consistent with the expected trend. After one hour, the cell density was highest for the cells exposed to 120,000 ppb; the cells with the galactose-induced promoter were higher than the negative control. After twenty hours of growth, the galactose-induced samples were higher in cell density than the glucose-induced samples in 40% of the concentrations (12 ppb and 120,000ppb).

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Ultimately, the test was inconclusive. We have two possible explanations for this failure. First, we hypothesize that there could be an error in preparing and/or aliquoting arsenate stock concentrations for the samples. For example, OD600 at 1200 ppb is consistently higher than 120 ppb and thus, may have a lower arsenate concentration than stated. Second, we observed 10-fold differences in the OD600 for some replicates, suggesting spillage could have happened when transferring cells from the shaker to the plate reader and vice-versa. Third, it is possible that a 3% glucose solution was insufficient to completely repress the promoter; as a result, PHO84, coding for increased arsenic absorption, was still overexpressed. For example, in the figure below, the galactose-induced and glucose-repressed samples followed similar values and trends over time.

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Stanford Location

Plasmid name: ACR1ACR2

DNA2.0 Gene #:

Organism: Saccharomyces cerevisiae

Device type: actuator

Barcode #’s of glycerol stocks: 0133027024

Box label: BIOE44 F15 Section #2 Box

Plasmid name: PHO84

DNA2.0 Gene #:

Organism: Saccharomyces cerevisiae

Device type: actuator

Barcode #’s of glycerol stocks: 0133027030

Box label: BIOE44 F15 Section #2 Box

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